Project Summary: Amyotrophic Lateral Sclerosis (ALS), the most common motor neuron disorder, is a fatal, neurodegenerative disease with a median survival time of 2-3 years from symptom onset. There are currently only two approved therapies for ALS, RilutekR, a channel modulator, and more recently, RadicavaR, a neuroprotective antioxidant. Neither of these therapies can halt disease progression, and ALS remains a devastating, fatal disease and major area of unmet medical need. Recently, there has been substantial progress in identifying ALS disease genes. This progress has helped promote understanding of processes in motor neurons that are affected by the disease. Despite these advances, many identified ALS genes do not encode traditional ?druggable? targets, and it has been challenging to translate these advances into new small molecule drugs for ALS. However, some of the ALS genes are good candidates for new, gene-based treatments, such as gene therapy or antisense oligonucleotide (ASO) therapy. One ALS gene for which there is strong rationale for a gene-based therapeutic approach is called FUS (Fused in Sarcoma). Mutations in FUS were first described in families afflicted with ALS in 2009. Since then, mutations in FUS were identified as one of the most common risk factors for juvenile onset ALS with particularly aggressive progression that typically results in death within one year of onset. There is strong evidence that mutations in FUS result in a toxic gene product. This evidence suggests that therapeutic approaches that can reduce the level of the mutant, toxic FUS product comprise a compelling basis for treating ALS patients with FUS mutations. This therapeutic mechanism could be achieved with an ASO designed to specifically reduce the FUS gene product made from the mutant copy of the FUS gene. ASOs are short, synthetic stretches of modified genetic material that can be designed to recognize and knockdown specific gene products. The recent approval of the ASO therapeutic, SpinrazaR, for spinal muscular atrophy has paved the way for ASO therapeutics for degenerative, neurological disorders. These ASO drugs are administered directly into the central nervous system by injection into the fluid surrounding the spinal cord. While therapeutic ASOs that target both gene copies are advancing in clinical trials for Huntington?s disease and SOD1-ALS, it has been more challenging to design ASOs that specifically target the mutant gene copies. Such ?allele-specific? ASOs would be ideal therapeutic candidates for many severe genetic neurological diseases. In this research program, we will validate a new design method for allele-specific ASOs. We will design and test allele-specific ASOs for multiple targets in the FUS gene that would enable specific knockdown of mutant FUS gene product for many ALS patients with FUS mutations. We will demonstrate that the ASOs achieve a high degree of allele specificity and are able to rescue a disease signature in a cell-based model of FUS-ALS. In subsequent phases of the research program, these candidate ASOs will be optimized and ultimately developed as treatments for ALS patients with FUS mutations. The validated allele-specific ASO technology would have significant medical and commercial value. Ultimately, the proposed research stands to benefit the population of ALS patients with FUS mutations, who currently suffer from a devastating, incurable and lethal condition, as well as patients suffering from other severe genetic diseases with toxic gain-of-function or dominant-negative disease mechanisms.